Binding of beta2 integrin LFA-1 to ICAM is sufficient to induce granule polarization but not degranulation, whereas CD16 binding to IgG triggers unpolarized degranulation. We have investigated the basis for this difference. IL-2 expanded human NK cells were stimulated by incubation with plate-bound ligands of LFA-1 (ICAM-1) and CD16 (human IgG). Surprisingly, LFA-1 elicited signals similar to those induced by CD16, including tyrosine phosphorylation of the TCR zeta chain, tyrosine kinase Syk, and phospholipase C (PLC)-gamma. Whereas CD16 activated Ca2+ mobilization and LAT phosphorylation, LFA-1 did not, but induced strong Pyk2 and paxillin phosphorylation. LFA-1-dependent granule polarization was blocked by inhibition of Syk, PLC-gamma, and PKC, and by paxillin knockdown. Therefore, common signals triggered by CD16 and LFA-1 bifurcate to provide independent control of Ca2+-dependent degranulation and paxillin-dependent granule polarization. To further explore the pathway by which LFA-1 signals in NK cells to induce granule and MTOC polarization toward the immunological synapse, we have employed a mass spectrometry approach. Primary NK cells stimulated by purified, recombinant ICAM-1 bound to plates were lysed, and tyrosine-phosphorylated proteins were isolated with a phosphotyrosine-specific antibody. As control, NK cells were also added to plates without ICAM-1. For comparison, NK cells were similarly stimulated by CD16 on plates carrying human IgG1. Mass spectrometry was then applied to sequence all the proteins isolated in this manner, which includes tyrosine-phosphorylated proteins and proteins bound to complexes that include tyrosine-phosphorylated proteins. This approach identified many proteins, some of which were enriched specifically in the ICAM-1-stimulated NK cells. The approach was validated by the presence of several proteins that had already been identified as undergoing tyrosine phosphorylation, such as Syk, PLC-gamma, paxillin, and p130CAS. An additional set of proteins, was identified, which was specific to stimulation through LFA-1. Several proteins that were enriched after stimulation by LFA-1 but not CD16 were validated by immunoblotting and their importance in LFA-1 signaling for granule polarization was tested by siRNA-mediated silencing. Integrin-linked kinase (ILK), gamma-parvin and RhoGEF7, all known to be involved in establishing cell polarity during migration, were critical for LFA-1-dependent granule polarization. Downstream signals for polarity in migrating cells require Par6 and phosphorylation of kinase GSK3beta at Ser9, which relieves inhibition of the microtubule-stabilizing molecule APC. Par6 and APC silencing blocked granule polarization. Binding to ICAM-1 alone induced an ILK-dependent GSK3beta Ser9 phosphorylation. Therefore, granule polarization induced by LFA-1 in NK cells uses a signaling pathway similar to that used to establish polarity during migration of adherent cells.